1. Field of the Invention
The present invention relates to the area of diphenyloxazole derivates, the synthesis thereof, and the use thereof as fluorescence dyes and probes. The dyes of the present invention are derivatives of 2-(4′-sulfamoylphenyl)-5-(4″-dimethylaminophenyl)oxazoles.
2. Related Art
Fluorescence is the result of a three-stage process that occurs when certain molecules absorb energy. The three stages comprise: 1) excitation; 2) excited-state lifetime; and 3) fluorescence emission. During stage 1, excitation, a photon of a certain energy is absorbed by the fluorophore. The fluorophore is initially in its ground state (S0). Absorption of the photon causes that fluorophore to become excited. The energy of the absorbed photon is transferred to an electron. The electron is transferred to a higher energy state. The fluorophore exists in an excited electronic singlet state (S1′), also called an excited state. The excited state of the fluorophore exists for a finite time, typically 10−8 to 10−9 seconds. During the excited state, the fluorophore changes in its translational, vibrational, and electronic energy states, and is subject to interactions with its molecular environment. The excited fluorophore releases energy and returns to the ground state, S0, by fluorescence emission. Other processes such as fluorescence energy transfer, intersystem crossing, and collisional quenching may also depopulate S1. The ratio of the number of fluorescence photons emitted, during the emission stage, to the number of photons absorbed, during the excitation stage, is termed the quantum yield. The quantum yield is a measure of the efficiency of fluorescence in competition with other processes such as fluorescence energy transfer, intersystem crossing, and collisional quenching.
During the third stage, fluorescence emission, a photon of energy hv (where h is Planck's constant and v is the frequency of the photon) is emitted, returning the fluorophore to its ground state S0. The energy of the emitted photon is lower than the energy of the photon absorbed during the excitation stage. The difference in energy can be attributed to dissipation through processes during the excited-state lifetime, such processes include fluorescence energy transfer, intersystem crossing, and collisional quenching. The difference in energy of the absorbed photon and the emitted photon is called the Stokes shift. The Stokes shift is fundamental to the sensitivity of fluorescence techniques because it allows emission photons to be detected against a low background, and at a different wavelength than the excitation photons.
Compounds that have fluorescent properties have numerous uses. Fluorescent molecules can be used in single molecule spectroscopy. Fluorescent molecules whose spectra or quantum yields are sensitive to their environments are valuable in the study of heterogeneous media, organized media, and biological media and many fluorescent dyes have been developed for these applications. However, many dyes either have short absorption and emission wavelengths (potentially causing high background due to the auto fluorescence of samples), low extinction coefficients, low quantum yields, or small Stokes shifts.
Fluorescent molecules are also useful in microplate thermal shift assays, as described in U.S. Pat. No. 6,020,141, which is fully incorporated by reference herein.
Rapid, high-throughput screening using fluorescence methodologies would also be facilitated by the use of fluorescence probe molecules that fluoresce at wavelengths longer than fluorescence molecules such as 1-anilinonaphthalene-8-sulfonate. That is because many molecules in compound and combinatorial libraries fluoresce at the same wavelengths at which presently available fluorescence probe molecules fluoresce. In addition, plastic microplates used in high-throughput screening assays may also fluoresce at the same wavelengths at which fluorescence probe molecules fluoresce.
Thus, there is a need for molecules that fluoresce when excited and provide emission spectra more useful than the spectra of 1-anilinonaph-thalene-8-sulfonate and derivatives thereof.
One class of fluorescent molecules is a group of compounds termed DAPOXYL dyes. DAPOXYL dyes contain the 4-(4′-(dimethylamino)phenyl)-2-(4′-sulfonylphenyl)oxazole moiety. A number of DAPOXYL dyes are known, including Dapoxyl® sulfonyl chloride; Dapoxy® carboxylic acid, succinimidyl ester; Dapoxyl® 3-sulfonamidopropionic acid, succinimidyl ester; Dapoxyl® (2-bromoacetamidoethyl)sulfonamide; Dapoxyl® 2-(3-(2-pyridyldithio)-propionamidoethyl) sulfonamide; Dapoxyl®) 3-sulfonamidophenylboronic acid; Dapoxyl® sulfonyl hydrazine; Dapoxyl® (2-aminoethyl)sulfonamide; Dapoxyl® sulfonic acid, sodium salt; and Dapoxyl® butylsulfonamide.
However, new derivatives of 2-(4′-sulfamoylphenyl)-5-(4′-dimethylaminophenyl)oxazole derivatives are needed that have improved solubility in both organic and aqueous media. New derivatives that are either more polar or less polar than existing oxazole dyes are needed. New oxazole derivatives that have improved utility in thermal shift assays are also needed.